In all tissues of the body there is a sub-population of adult stem cells. These multipotent cells are recruited and activated to take part in tissue regeneration. Adult stem cells are a promising resource for therapy, but their numbers are very low and they need propagation in vitro to be of therapeutic use. When these cells are cultured ex-vivo it has proven difficult to recreate their natural microenvironment, which is thought to be a sum of signals from interactions with the extracellular matrix and neighboring cells and the hormonal status of the microenvironment. Therefore, regenerative therapies using adult stem cells are still hampered by the limited number of available cells and the fact that their expansion in vitro, necessary to attain therapeutic numbers, compromises their differentiation and proliferative potential.
Due to their capacity to form cartilage, bone, fat and other connective tissue, human mesenchymal stem cells (hMSCs) constitute an exciting prospect for cell-based therapy in regenerating diseased or injured tissues. These adult stem cells can be readily purified from a small volume of bone marrow aspirates, and expanded in vitro for a limited number of population doublings (PD) (=30) before they reach replicative senescence. It is likely that this growth arrest is linked to telomere shortening as over-expression of the catalytic subunit of the telomerase (hTERT) is sufficient to increase the life span to several hundred population doublings. These “telomerized” cells retain their ability to assume phenotypes of mesenchymal tissues, thus providing a useful tool for the study of hMSCs. However, it does not address the issue of attaining a therapeutic number of multipotent stem cells in culture without severely affecting their regenerative potential.
The spontaneous differentiation of stem cells in culture is a result of a change in the microenvironment from that normally found in the naive stem cell niche. As mentioned above, the stem cell niche is a sum of signals from interactions with specific components of the extracellular matrix (ECM) and neighboring cells, and the hormonal status of the microenvironment.
Thus, there exists a need for methods and media compositions that help to overcome the problems encountered in the expansion of ex vivo stem cell cultures.
The capacity of adult human stem cells for both self-renewal and directed differentiation is efficacious for cell-based therapy, with bone marrow-derived human mesenchymal stem cells (hMSCs) representing one of the few stem cell types currently in clinical trials1. Tissue regeneration has been reported after delivery of adult stem cells either locally or systemically2-5. Moreover, hMSCs have shown potential for cardiovascular regeneration6 and are currently undergoing phase III clinical trials. These cells have also shown potent immunosuppressive effects in vivo7, making them particularly suited to transplantation. Despite such promise, widespread use of hMSCs is hindered by their low abundance (<0.01% of bone marrow mononuclear cells, BMMNCs)8. Successful enrichment of hMSCs using the monoclonal antibody STRO-19 is possible, although to reach sufficient numbers for therapy these cells require further ex vivo expansion. Although some ex vivo expansion is possible, a loss of multipotentiality occurs within a relatively short period of time10. Conditions that mimic the bone marrow microenvironment and maximize hMSC proliferation without adversely affecting multipotentiality (“stemness”) are therefore needed.
Several strategies have been developed for the ex vivo expansion of hMSCs, including the forced expression of hTERT (telomerase catalytic sub-unit)11,12, the addition of soluble peptide mitogens13-18, and the use of extracellular matrix (ECM) molecules19-21. When transduced with hTERT, hMSCs fail to senesce and can be cultured for more than 260 population doublings. However these cells become tumorigenic22, making this strategy untenable. The addition of growth factors, particularly fibroblast growth factor-2 (FGF-2), has also been shown to increase hMSC expansion. However it also increases the proliferation of more differentiated cells18. Key elements of the ECM are also known to support stem cell self-renewal, and strategies that manipulate them have shown promise20. One of the most active ECM species contributing to improved growth is the family of heparan sulphate (also called heparan sulphate or HS) glycosaminoglycan (GAG) sugars23,24; the actions of many growth factors are known to be dependent on specific forms of this carbohydrate.
A major challenge for hMSC therapy is the provision of therapeutic numbers of multipotent stem cells. Current strategies utilized for generating hMSCs for clinical use rely on their isolation by adherence to plastic, followed by lengthy ex vivo expansion prior to re-implantation. However, many mesenchymal stem cells remain quiescent when isolated from adult bone marrow and cultured ex vivo, and will therefore fail to proliferate.